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1.
Phys Rev E ; 103(3-1): 032409, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33862816

RESUMO

The mechanical behavior and cortical tension of single cells are analyzed using electrodeformation relaxation. Four types of cells, namely, MCF-10A, MCF-7, MDA-MB-231, and GBM, are studied, with pulse durations ranging from 0.01 to 10 s. Mechanical response in the long-pulse regime is characterized by a power-law behavior, consistent with soft glassy rheology resulting from unbinding events within the cortex network. In the subsecond short-pulse regime, a single timescale well describes the process and indicates the naive tensioned (prestressed) state of the cortex with minimal force-induced alteration. A mathematical model is employed and the simple ellipsoidal geometry allows for use of an analytical solution to extract the cortical tension. At the shortest pulse of 0.01 s, tensions for all four cell types are on the order of 10^{-2} N/m.


Assuntos
Fenômenos Mecânicos , Análise de Célula Única , Modelos Teóricos , Reologia
2.
Biophys J ; 114(11): 2703-2716, 2018 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-29874619

RESUMO

Multicellular aggregates are an excellent model system to explore the role of tissue biomechanics in specifying multicellular reorganization during embryonic developments and malignant invasion. Tissue-like spheroids, when subjected to a compressive force, are known to exhibit liquid-like behaviors at long timescales (hours), largely because of cell rearrangements that serve to effectively dissipate the applied stress. At short timescales (seconds to minutes), before cell rearrangement, the mechanical behavior is strikingly different. The current work uses shape relaxation to investigate the structural characteristics of aggregates and discovers two coherent timescales: one on the order of seconds, the other tens of seconds. These timescales are universal, conserved across a variety of tested species, and persist despite great differences in other properties such as tissue surface tension and adhesion. A precise mathematical theory is used to correlate the timescales with mechanical properties and reveals that aggregates have a relatively strong envelope and an unusually "soft" interior (weak bulk elastic modulus). This characteristic is peculiar, considering that both layers consist of identical units (cells), but is consistent with the fact that this structure can engender both structural integrity and the flexibility required for remodeling. In addition, tissue surface tension, elastic modulus, and viscosity are proportional to each other. Considering that these tissue-level properties intrinsically derive from cellular-level properties, the proportionalities imply precise coregulation of the latter and in particular of the tension on the cell-medium and cell-cell interfaces.


Assuntos
Agregação Celular , Fenômenos Mecânicos , Fenômenos Biomecânicos , Linhagem Celular , Humanos , Modelos Biológicos , Esferoides Celulares/citologia
3.
Int J Mol Sci ; 19(2)2018 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-29443896

RESUMO

Dispersal of Glioblastoma (GBM) renders localized therapy ineffective and is a major cause of recurrence. Previous studies have demonstrated that Dexamethasone (Dex), a drug currently used to treat brain tumor-related edema, can also significantly reduce dispersal of human primary GBM cells from neurospheres. It does so by triggering α5 integrin activity, leading to restoration of fibronectin matrix assembly (FNMA), increased neurosphere cohesion, and reduction of neurosphere dispersal velocity (DV). How Dex specifically activates α5 integrin in these GBM lines is unknown. Several chaperone proteins are known to activate integrins, including calreticulin (CALR). We explore the role of CALR as a potential mediator of Dex-dependent induction of α5 integrin activity in primary human GBM cells. We use CALR knock-down and knock-in strategies to explore the effects on FNMA, aggregate compaction, and dispersal velocity in vitro, as well as dispersal ex vivo on extirpated mouse retina and brain slices. We show that Dex increases CALR expression and that siRNA knockdown suppresses Dex-mediated FNMA. Overexpression of CALR in GBM cells activates FNMA, increases compaction, and decreases DV in vitro and on explants of mouse retina and brain slices. Our results define a novel interaction between Dex, CALR, and FNMA as inhibitors of GBM dispersal.


Assuntos
Antineoplásicos Hormonais/farmacologia , Neoplasias Encefálicas/metabolismo , Calreticulina/genética , Dexametasona/farmacologia , Glioblastoma/metabolismo , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Calreticulina/metabolismo , Células Cultivadas , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Fibronectinas/metabolismo , Humanos , Camundongos , Retina/efeitos dos fármacos , Retina/metabolismo , Regulação para Cima
4.
PLoS One ; 12(10): e0186483, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29040322

RESUMO

Glioblastoma is highly aggressive. Early dispersal of the primary tumor renders localized therapy ineffective. Recurrence always occurs and leads to patient death. Prior studies have shown that dispersal of Glioblastoma can be significantly reduced by Dexamethasone (Dex), a drug currently used to control brain tumor related edema. However, due to high doses and significant side effects, treatment is tapered and discontinued as soon as edema has resolved. Prior analyses of the dispersal inhibitory effects of Dex were performed on tissue culture plastic, or polystyrene filters seeded with normal human astrocytes, conditions which inherently differ from the parenchymal architecture of neuronal tissue. The aim of this study was to utilize an ex-vivo model to examine Dex-mediated inhibition of tumor cell migration from low-passage, human Glioblastoma neurospheres on multiple substrates including mouse retina, and slices of mouse, pig, and human brain. We also determined the lowest possible Dex dose that can inhibit dispersal. Analysis by Two-Factor ANOVA shows that for GBM-2 and GBM-3, Dex treatment significantly reduces dispersal on all tissue types. However, the magnitude of the effect appears to be tissue-type specific. Moreover, there does not appear to be a difference in Dex-mediated inhibition of dispersal between mouse retina, mouse brain and human brain. To estimate the lowest possible dose at which Dex can inhibit dispersal, LogEC50 values were compared by Extra Sum-of-Squares F-test. We show that it is possible to achieve 50% reduction in dispersal with Dex doses ranging from 3.8 x10-8M to 8.0x10-9M for GBM-2, and 4.3x10-8M to 1.8x10-9M for GBM-3, on mouse retina and brain slices, respectively. These doses are 3-30-fold lower than those used to control edema. This study extends our previous in vitro data and identifies the mouse retina as a potential substrate for in vivo studies of GBM dispersal.


Assuntos
Antineoplásicos Hormonais/farmacologia , Movimento Celular/efeitos dos fármacos , Dexametasona/farmacologia , Neuroglia/efeitos dos fármacos , Esferoides Celulares/efeitos dos fármacos , Animais , Encéfalo/citologia , Relação Dose-Resposta a Droga , Células Alimentadoras/citologia , Humanos , Camundongos , Microtomia , Neuroglia/patologia , Retina/citologia , Esferoides Celulares/patologia , Suínos , Células Tumorais Cultivadas
5.
BMC Cancer ; 17(1): 121, 2017 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-28187762

RESUMO

BACKGROUND: Dispersal of glioblastoma (GBM) cells leads to recurrence and poor prognosis. Accordingly, molecular pathways involved in dispersal are potential therapeutic targets. The mitogen activated protein kinase/extracellular signal regulated kinase (MAPK/ERK) pathway is commonly dysregulated in GBM, and targeting this pathway with MEK inhibitors has proven effective in controlling tumor growth. Since this pathway also regulates ECM remodeling and actin organization - processes crucial to cell adhesion, substrate attachment, and cell motility - the aim of this study was to determine whether inhibiting this pathway could also impede dispersal. METHODS: A variety of methods were used to quantify the effects of the MEK inhibitor, PD0325901, on potential regulators of dispersal. Cohesion, stiffness and viscosity were quantified using a method based on ellipsoid relaxation after removal of a deforming external force. Attachment strength, cell motility, spheroid dispersal velocity, and 3D growth rate were quantified using previously described methods. RESULTS: We show that PD0325901 significantly increases aggregate cohesion, stiffness, and viscosity but only when tumor cells have access to high concentrations of fibronectin. Treatment also results in reorganization of actin from cortical into stress fibers, in both 2D and 3D culture. Moreover, drug treatment localized pFAK at sites of cell-substratum adhesion. Collectively, these changes resulted in increased strength of substrate attachment and decreased motility, a decrease in aggregate dispersal velocity, and in a marked decrease in growth rate of both 2D and 3D cultures. CONCLUSIONS: Inhibition of the MAPK/ERK pathway by PD0325901 may be an effective therapy for reducing dispersal and growth of GBM cells.


Assuntos
Benzamidas/farmacologia , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Difenilamina/análogos & derivados , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Actinas/metabolismo , Adesão Celular/efeitos dos fármacos , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Difenilamina/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Microscopia Confocal , Fosforilação/efeitos dos fármacos , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismo
6.
PLoS One ; 10(8): e0135951, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26284619

RESUMO

Despite resection and adjuvant therapy, the 5-year survival for patients with Glioblastoma multiforme (GBM) is less than 10%. This poor outcome is largely attributed to rapid tumor growth and early dispersal of cells, factors that contribute to a high recurrence rate and poor prognosis. An understanding of the cellular and molecular machinery that drive growth and dispersal is essential if we are to impact long-term survival. Our previous studies utilizing a series of immortalized GBM cell lines established a functional causation between activation of fibronectin matrix assembly (FNMA), increased tumor cohesion, and decreased dispersal. Activation of FNMA was accomplished by treatment with Dexamethasone (Dex), a drug routinely used to treat brain tumor related edema. Here, we utilize a broad range of qualitative and quantitative assays and the use of a human GBM tissue microarray and freshly-isolated primary human GBM cells grown both as conventional 2D cultures and as 3D spheroids to explore the role of Dex and FNMA in modulating various parameters that can significantly influence tumor cell dispersal. We show that the expression and processing of fibronectin in a human GBM tissue-microarray is variable, with 90% of tumors displaying some abnormality or lack in capacity to secrete fibronectin or assemble it into a matrix. We also show that low-passage primary GBM cells vary in their capacity for FNMA and that Dex treatment reactivates this process. Activation of FNMA effectively "glues" cells together and prevents cells from detaching from the primary mass. Dex treatment also significantly increases the strength of cell-ECM adhesion and decreases motility. The combination of increased cohesion and decreased motility discourages in vitro and ex vivo dispersal. By increasing cell-cell cohesion, Dex also decreases growth rate of 3D spheroids. These effects could all be reversed by an inhibitor of FNMA and by the glucocorticoid receptor antagonist, RU-486. Our results describe a new role for Dex as a suppressor of GBM dispersal and growth.


Assuntos
Neoplasias Encefálicas/patologia , Adesão Celular/fisiologia , Agregação Celular/fisiologia , Dexametasona/farmacologia , Fibronectinas/metabolismo , Glioblastoma/patologia , Anti-Inflamatórios/farmacologia , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/metabolismo , Matriz Extracelular/metabolismo , Glioblastoma/tratamento farmacológico , Glioblastoma/metabolismo , Humanos , Integrina alfa5beta1/metabolismo , Invasividade Neoplásica , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Esferoides Celulares/metabolismo , Células Tumorais Cultivadas
7.
Methods Mol Biol ; 1189: 237-54, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25245698

RESUMO

Intercellular adhesion plays a vital role in many biological processes including embryonic development, malignant invasion, and wound healing, and can be manipulated to generate complex structures in tissue engineering applications. Accurate measurement of the strength of intercellular adhesion is not trivial and requires methods rooted in sound physical principles. Tissue surface tensiometry (TST) rigorously quantifies intercellular cohesive energy of 3D tissue-like aggregates under physiological conditions. TST utilizes a custom-built tensiometer to compress 3D spheroids between parallel plates. The resistance to the applied force and changes in aggregate geometry are applied to the Young-Laplace equation, generating a measurement of apparent surface tension. We describe all components comprising the tensiometer and provide step by step instructions of all the key steps involved in generating spherical aggregates. We explain how tissue surface tension is calculated and provide a statistical analysis of a sample data set from 12 aggregates.


Assuntos
Biofísica/métodos , Espaço Extracelular/metabolismo , Animais , Adesão Celular , Agregação Celular , Embrião de Galinha , Fígado/embriologia , Tensão Superficial
8.
Wiley Interdiscip Rev Dev Biol ; 2(5): 631-45, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24014451

RESUMO

During embryonic development, cells or groups of cells migrate from their locations of origin to assume their correct anatomical positions. Intercellular adhesion plays an active and instructive role in orchestrating this process. Precisely how adhesion provides spatial positioning information is a subject of intense interest. In the 1960s, Steinberg proposed the differential adhesion hypothesis (DAH) to explain how differences in the intensity of cell adhesion could give rise to predictable spatial interactions between different cell types. The DAH is grounded in the same set of physical principles governing the interaction of immiscible fluids and thus provides a rigorous conceptual framework connecting the chemistry of cell adhesion to the physics underlying cell and tissue segregation. Testing the DAH required the development of methods to measure intercellular cohesion and of assays to accurately assess relative spatial position between cells. The DAH has been experimentally verified and computationally simulated. Moreover, evidence concerning the role of differential adhesion in a number of morphodynamic events is accumulating. It is clear that differential adhesion is a major driving force in various aspects of embryonic development, but recent studies have also advanced the concept that other factors, such as cortical tension and elasticity, may also be involved in fine tuning, or even driving the process. It is likely that an interplay between adhesion and these other factors co-operate to generate the forces required for tissue self-organization.


Assuntos
Indução Embrionária , Animais , Adesão Celular , Camadas Germinativas/citologia , Camadas Germinativas/metabolismo , Camadas Germinativas/fisiologia , Humanos , Termodinâmica
9.
Future Oncol ; 9(8): 1121-32, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23902244

RESUMO

Patients with glioblastoma typically present when tumors are at an advanced stage. Surgical resection, radiotherapy and adjuvant chemotherapy are currently the standard of care for glioblastoma. However, due to the infiltrative and dispersive nature of the tumor, recurrence rate remains high and typically results in very poor prognosis. Efforts to treat the primary tumor are, therefore, palliative rather than curative. From a practical perspective, controlling growth and dispersal of the recurrence may have a greater impact on disease-free survival. In order for cells to disperse, they must first detach from the mass. Preventing detachment may keep tumors that recur more localized and perhaps more amenable to therapy. Here we introduce a new perspective in which a quantifiable mechanical property, namely tissue surface tension, can provide novel information on tumor behavior. The overall theme of the discussion will attempt to integrate how adhesion molecules can alter a tumor's mechanical properties and how, in turn, these properties can be modified to prevent tumor cell detachment and dispersal.


Assuntos
Neoplasias Encefálicas/genética , Adesão Celular/genética , Glioblastoma/genética , Integrinas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/terapia , Linhagem Celular Tumoral , Intervalo Livre de Doença , Matriz Extracelular/metabolismo , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Integrinas/metabolismo , Modelos Teóricos , Terapia de Alvo Molecular , Recidiva Local de Neoplasia/metabolismo , Recidiva Local de Neoplasia/patologia , Recidiva Local de Neoplasia/terapia , Estadiamento de Neoplasias , Prognóstico
10.
BMC Cancer ; 12: 94, 2012 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-22433434

RESUMO

BACKGROUND: Invasion is an important early step in the metastatic cascade and is the primary cause of death of prostate cancer patients. In order to invade, cells must detach from the primary tumor. Cell-cell and cell-ECM interactions are important regulators of cohesion--a property previously demonstrated to mediate cell detachment and invasion. The studies reported here propose a novel role for α5ß1 integrin--the principle mediator of fibronectin matrix assembly (FNMA)--as an invasion suppressor of prostate cancer cells. METHODS: Using a combination of biophysical and cell biological methods, and well-characterized prostate cancer cell lines of varying invasiveness, we explore the relationship between cohesion, invasiveness, and FNMA. RESULTS: We show that cohesion is inversely proportional to invasive capacity. We also show that more invasive cells express lower levels of α5ß1 integrin and lack the capacity for FNMA. Cells were generated to over-express either wild-type α5 integrin or an integrin in which the cytoplasmic domain of α5 was replaced with that of α2. The α2 construct does not promote FNMA. We show that only wild-type α5 integrin promotes aggregate compaction, increases cohesion, and reduces invasion of the more aggressive cells, and that these effects can be blocked by the 70-kDa fibronectin fragment. CONCLUSIONS: We propose that restoring capacity for FNMA in deficient cells can increase tumor intercellular cohesion to a point that significantly reduces cell detachment and subsequent invasion. In prostate cancer, this could be of therapeutic benefit by blocking an early key step in the metastatic cascade.


Assuntos
Adesão Celular/fisiologia , Matriz Extracelular/metabolismo , Fibronectinas/metabolismo , Neoplasias da Próstata/metabolismo , Análise de Variância , Agregação Celular/fisiologia , Linhagem Celular Tumoral , Humanos , Integrina alfa5beta1/metabolismo , Masculino , Invasividade Neoplásica , Neoplasias da Próstata/patologia , Tensão Superficial
11.
PLoS One ; 6(9): e24810, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21980357

RESUMO

Glioblastoma (GBM), the most aggressive and most common form of primary brain tumor, has a median survival of 12-15 months. Surgical excision, radiation and chemotherapy are rarely curative since tumor cells broadly disperse within the brain. Preventing dispersal could be of therapeutic benefit. Previous studies have reported that increased cell-cell cohesion can markedly reduce invasion by discouraging cell detachment from the tumor mass. We have previously reported that α5ß1 integrin-fibronectin interaction is a powerful mediator of indirect cell-cell cohesion and that the process of fibronectin matrix assembly (FNMA) is crucial to establishing strong bonds between cells in 3D tumor-like spheroids. Here, we explore a potential role for FNMA in preventing dispersal of GBM cells from a tumor-like mass. Using a series of GBM-derived cell lines we developed an in vitro assay to measure the dispersal velocity of aggregates on a solid substrate. Despite their similar pathologic grade, aggregates from these lines spread at markedly different rates. Spreading velocity is inversely proportional to capacity for FNMA and restoring FNMA in GBM cells markedly reduces spreading velocity by keeping cells more connected. Blocking FNMA using the 70 KDa fibronectin fragment in FNMA-restored cells rescues spreading velocity, establishing a functional role for FNMA in mediating dispersal. Collectively, the data support a functional causation between restoration of FNMA and decreased dispersal velocity. This is a first demonstration that FNMA can play a suppressive role in GBM dispersal.


Assuntos
Neoplasias Encefálicas/metabolismo , Fibronectinas/metabolismo , Glioblastoma/metabolismo , Caderinas/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Dexametasona/farmacologia , Regulação Neoplásica da Expressão Gênica , Humanos , Integrina alfa5beta1/metabolismo , Microscopia de Fluorescência/métodos , Modelos Estatísticos , Análise de Regressão , Esferoides Celulares/metabolismo
12.
Biochemistry ; 50(20): 4309-21, 2011 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-21491912

RESUMO

Proteins encoded by the epidermal growth factor receptor (EGFR/HER1/ERBB1) gene are being studied as diagnostic, prognostic, and theragnostic biomarkers for numerous human cancers. The clinical application of these tissue/tumor biomarkers has been limited, in part, by discordant results observed for epidermal growth factor receptor (EGFR) expression using different immunological reagents. Previous studies have used EGFR-directed antibodies that cannot distinguish between full-length and soluble EGFR (sEGFR) expression. We have generated and characterized an anti-sEGFR polyclonal antiserum directed against a 31-mer peptide (residues 604-634) located within the unique 78-amino acid carboxy-terminal sequence of sEGFR. Here, we use this antibody to demonstrate that sEGFR is coexpressed with EGFR in a number of carcinoma-derived cell lines. In addition, we show that a second protein of ~140 kDa (p140) also is detected by this antibody. Rigorous biochemical characterization identifies this second protein to be α5-integrin. We show that a 26-amino acid peptide in the calf domain of α5-integrin (residues 710-735) is 35% identical in sequence with a 31-mer carboxy-terminal sEGFR peptide and exhibits an approximately 5-fold lower affinity for anti-sEGFR than the homologous 31-mer sEGFR peptide does. We conclude that the carboxy terminus of sEGFR and the calf-1 domain of α5-integrin share a region of sequence identity, which results in their mutual immunological reactivity with anti-sEGFR. We also demonstrate that anti-sEGFR promotes three-dimensional tissue cohesion and compaction in vitro, further suggesting a functional link between sEGFR and α5-integrin and a role of the calf-1 domain in cell adhesion. These results have implications for the study of both EGFR and sEGFR as cancer biomarkers and also provide new insight into the mechanisms of interaction between cell surface EGFR isoforms and integrins in complex processes such as cell adhesion and survival signaling.


Assuntos
Mapeamento de Epitopos/métodos , Epitopos/imunologia , Receptores ErbB/química , Receptores ErbB/imunologia , Integrina alfa5/química , Integrina alfa5/imunologia , Homologia de Sequência de Aminoácidos , Sequência de Aminoácidos , Animais , Anticorpos/imunologia , Afinidade de Anticorpos , Células CHO , Agregação Celular , Linhagem Celular Tumoral , Sequência Conservada , Cricetinae , Cricetulus , Detergentes/química , Humanos , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Solubilidade
13.
J Vis Exp ; (50)2011 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-21505411

RESUMO

Rigorous measurement of intercellular binding energy can only be made using methods grounded in thermodynamic principles in systems at equilibrium. We have developed tissue surface tensiometry (TST) specifically to measure the surface free energy of interaction between cells. The biophysical concepts underlying TST have been previously described in detail. The method is based on the observation that mutually cohesive cells, if maintained in shaking culture, will spontaneously assemble into clusters. Over time, these clusters will round up to form spheres. This rounding-up behavior mimics the behavior characteristic of liquid systems. Intercellular binding energy is measured by compressing spherical aggregates between parallel plates in a custom-designed tissue surface tensiometer. The same mathematical equation used to measure the surface tension of a liquid droplet is used to measure surface tension of 3D tissue-like spherical aggregates. The cellular equivalent of liquid surface tension is intercellular binding energy, or more generally, tissue cohesivity. Previous studies from our laboratory have shown that tissue surface tension (1) predicts how two groups of embryonic cells will interact with one another, (2) can strongly influence the ability of tissues to interact with biomaterials, (3) can be altered not only through direct manipulation of cadherin-based intercellular cohesion, but also by manipulation of key ECM molecules such as FN and 4) correlates with invasive potential of lung cancer, fibrosarcoma, brain tumor and prostate tumor cell lines. In this article we will describe the apparatus, detail the steps required to generate spheroids, to load the spheroids into the tensiometer chamber, to initiate aggregate compression, and to analyze and validate the tissue surface tension measurements generated.


Assuntos
Adesão Celular/fisiologia , Comunicação Celular/fisiologia , Técnicas Citológicas/instrumentação , Técnicas Citológicas/métodos , Animais , Fibroblastos/citologia , Masculino , Músculo Liso/citologia , Próstata/citologia , Ratos , Tensão Superficial
14.
Am J Respir Cell Mol Biol ; 44(5): 682-91, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-20616358

RESUMO

To identify cell-intrinsic properties that facilitate interaction between epithelial endodermal and mesenchymal mesodermal cells during lung morphogenesis, we developed a model of lung self-assembly that mimics fetal lung formation in structure, polarity, vasculature, and extracellular matrix expression. Three-dimensional pulmonary bodies (PBs) spontaneously self-assemble from single-cell suspensions and exhibit liquid-like properties that allow measurements of compaction rate and cohesion, and that may help to specify cellular self-organization. We hypothesized that changes in one or more of these parameters could potentially explain the lung hypoplasia associated with abnormal lung development. We examined the impact of endothelial/monocyte-activating polypeptide (EMAP) II in PBs, because EMAPII is highly expressed in lung hypoplasia. EMAPII significantly increased compaction rate and decreased overall cohesion of PBs composed of both epithelial and mesenchymal cells. Moreover, the effects of EMAPII on compaction and cohesion act exclusively through the mesenchymal cell population by interfering with fibronectin matrix assembly. We also show that EMAPII alters epithelial cell polarity and surfactant protein C expression. Our findings demonstrate, for the first time, that PBs possess liquid-like properties that can help to guide the self-assembly of fetal lungs, and that EMAPII expression can influence both mesenchymal and epithelial cells but through different molecular mechanisms.


Assuntos
Pulmão/fisiologia , Animais , Células CHO , Comunicação Celular , Cricetinae , Cricetulus , Citocinas/metabolismo , Células Epiteliais/citologia , Matriz Extracelular/metabolismo , Fibronectinas/metabolismo , Regulação da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intercelular , Pulmão/embriologia , Pulmão/metabolismo , Células-Tronco Mesenquimais/citologia , Camundongos , Proteínas de Neoplasias/metabolismo , Peptídeos/metabolismo , Proteína C Associada a Surfactante Pulmonar , Proteínas de Ligação a RNA/metabolismo , Tensão Superficial
15.
PLoS One ; 5(7): e11830, 2010 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-20686611

RESUMO

BACKGROUND: Tissue organization during embryonic development and wound healing depends on the ability of cells on the one hand to exchange adhesive bonds during active rearrangement and on the other to become fixed in place as tissue homeostasis is reached. Cells achieve these contradictory tasks by regulating either cell-cell adhesive bonds, mediated by cadherins, or cell-extracellular matrix (ECM) connections, regulated by integrins. Integrin alpha5beta1 and soluble fibronectin (sFN) are key players in cell-ECM force generation and in ECM polymerization. Here, we explore the interplay between integrin alpha5beta1 and sFN and its influence on tissue mechanical properties and cell sorting behavior. METHODOLOGY/PRINCIPAL FINDINGS: We generated a series of cell lines varying in alpha5beta1 receptor density. We then systematically explored the effects of different sFN concentrations on aggregate biomechanical properties using tissue surface tensiometry. We found previously unreported complex behaviors including the observation that interactions between fibronectin and integrin alpha5beta1 generates biphasic tissue cohesion profiles. Specifically, we show that at constant sFn concentration, aggregate cohesion increases linearly as alpha5beta1 receptor density is increased from low to moderate levels, producing a transition from viscoelastic-liquid to pseudo viscoelastic-solid behavior. However, further increase in receptor density causes an abrupt drop in tissue cohesion and a transition back to viscoelastic-liquid properties. We propose that this may be due to depletion of sFn below a critical value in the aggregate microenvironment at high alpha5beta1 levels. We also show that differential expression of alpha5beta1 integrin can promote phase-separation between cells. CONCLUSIONS/SIGNIFICANCE: The interplay between alpha5-integrin and sFn contributes significantly to tissue cohesion and, depending on their level of expression, can mediate a shift from liquid to elastic behavior. This interplay represents a tunable level of control between integrins and the ECM that can influence tissue cohesion and other mechanical properties, which may translate to the specification of tissue structure and function. These studies provide insights into important biological processes such as embryonic development, wound healing, and for tissue engineering applications.


Assuntos
Fibronectinas/metabolismo , Integrina alfa5beta1/metabolismo , Animais , Fenômenos Biomecânicos , Células CHO , Cricetinae , Cricetulus , Ensaio de Imunoadsorção Enzimática , Matriz Extracelular/metabolismo , Citometria de Fluxo , Humanos , Microscopia de Fluorescência , Ligação Proteica
16.
Proc Natl Acad Sci U S A ; 107(28): 12517-22, 2010 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-20616053

RESUMO

In the course of animal morphogenesis, large-scale cell movements occur, which involve the rearrangement, mutual spreading, and compartmentalization of cell populations in specific configurations. Morphogenetic cell rearrangements such as cell sorting and mutual tissue spreading have been compared with the behaviors of immiscible liquids, which they closely resemble. Based on this similarity, it has been proposed that tissues behave as liquids and possess a characteristic surface tension, which arises as a collective, macroscopic property of groups of mobile, cohering cells. But how are tissue surface tensions generated? Different theories have been proposed to explain how mesoscopic cell properties such as cell-cell adhesion and contractility of cell interfaces may underlie tissue surface tensions. Although recent work suggests that both may be contributors, an explicit model for the dependence of tissue surface tension on these mesoscopic parameters has been missing. Here we show explicitly that the ratio of adhesion to cortical tension determines tissue surface tension. Our minimal model successfully explains the available experimental data and makes predictions, based on the feedback between mechanical energy and geometry, about the shapes of aggregate surface cells, which we verify experimentally. This model indicates that there is a crossover from adhesion dominated to cortical-tension dominated behavior as a function of the ratio between these two quantities.


Assuntos
Fenômenos Físicos , Animais , Adesão Celular , Movimento Celular , Separação Celular , Córtex Cerebral , Morfogênese , Tensão Superficial , Aderências Teciduais , Peixe-Zebra
17.
HFSP J ; 2(1): 42-56, 2008 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19404452

RESUMO

This study provides direct functional evidence that differential adhesion, measurable as quantitative differences in tissue surface tension, influences spatial positioning between zebrafish germ layer tissues. We show that embryonic ectodermal and mesendodermal tissues generated by mRNA-overexpression behave on long-time scales like immiscible fluids. When mixed in hanging drop culture, their cells segregate into discrete phases with ectoderm adopting an internal position relative to the mesendoderm. The position adopted directly correlates with differences in tissue surface tension. We also show that germ layer tissues from untreated embryos, when extirpated and placed in culture, adopt a configuration similar to those of their mRNA-overexpressing counterparts. Down-regulating E-cadherin expression in the ectoderm leads to reduced surface tension and results in phase reversal with E-cadherin-depleted ectoderm cells now adopting an external position relative to the mesendoderm. These results show that in vitro cell sorting of zebrafish mesendoderm and ectoderm tissues is specified by tissue interfacial tensions. We perform a mathematical analysis indicating that tissue interfacial tension between actively motile cells contributes to the spatial organization and dynamics of these zebrafish germ layers in vivo.

18.
Dev Dyn ; 236(8): 2039-49, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17584863

RESUMO

The organization of endocrine cells in pancreatic islets is established through a series of morphogenetic events involving cell sorting, migration, and re-aggregation processes for which intercellular adhesion is thought to play a central role. In animals, these morphogenetic events result in an islet topology in which insulin-secreting cells form the core, while glucagon, somatostatin, and pancreatic polypeptide-secreting cells segregate to the periphery. Isolated pancreatic islet cells self-assemble in vitro into pseudoislets with the same cell type organization as native islets. It is widely held that differential adhesion between cells of the pancreatic islets generates this specific topology. However, this differential adhesion has never been rigorously quantified. In this manuscript, we use tissue surface tensiometry to measure the cohesivity of spherical aggregates from three immortalized mouse pancreatic islet cell lines. We show that, as predicted by the differential adhesion hypothesis, aggregates of the internally segregating INS-1 and MIN6 beta-cell lines are substantially more cohesive than those of the externally segregating alpha-TC line. Furthermore, we show that forced overexpression of P-cadherin by alpha-TC cells significantly perturbs the sorting process. Collectively, the data indicate that differential adhesion can drive the in vitro organization of immortalized rodent pancreatic islet cells.


Assuntos
Ilhotas Pancreáticas/citologia , Morfogênese , Animais , Caderinas/genética , Caderinas/farmacologia , Adesão Celular , Linhagem Celular , Técnicas de Cocultura , Camundongos , Modelos Biológicos , Tensão Superficial
19.
Bioessays ; 28(8): 809-21, 2006 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16927301

RESUMO

Throughout embryonic development, segregated epithelial and/or mesenchymal cell populations make contact and fuse to shape new tissue units. This process, known as tissue fusion, is a key event in many essential morphogenetic mechanisms and its disruption can lead to congenital malformations. Another mechanism whereby complex tissues can arise involves a cell sorting process in which originally intermixed cells de-mix to generate distinct phases or layers. Different organisms use a combination of tissue fusion and cell sorting to acquire shape. Although the two processes appear to differ mechanistically, they are intricately linked inasmuch as they both involve the same molecular determinants and contribute to the same body plan. We aim to discuss the role of adhesion molecules and cell dynamics in tissue fusion and cell sorting, providing examples of their impact in embryonic development. Finally, we will advance the concept that malignant invasion may be viewed as cell sorting in reverse. Supplementary material for this article can be found on the BioEssays website (http://www.interscience.wiley.com/jpages/0265-9247/suppmat/index.html).


Assuntos
Fusão Celular , Desenvolvimento Embrionário , Animais , Padronização Corporal , Adesão Celular , Desenvolvimento Embrionário/fisiologia , Humanos , Integrinas/fisiologia , Modelos Biológicos , Neoplasias/etiologia , Neoplasias/patologia
20.
Dev Biol ; 278(1): 255-63, 2005 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-15649477

RESUMO

The differential adhesion hypothesis (DAH), advanced in the 1960s, proposed that the liquid-like tissue-spreading and cell segregation phenomena of development arise from tissue surface tensions that in turn arise from differences in intercellular adhesiveness. Our earlier measurements of liquid-like cell aggregate surface tensions have shown that, without exception, a cell aggregate of lower surface tension tends to envelop one of higher surface tension to which it adheres. We here measure the surface tensions of L cell aggregates transfected to express N-, P- or E-cadherin in varied, measured amounts. We report that in these aggregates, in which cadherins are essentially the only cell-cell adhesion molecules, the aggregate surface tensions are a direct, linear function of cadherin expression level. Taken together with our earlier results, the conclusion follows that the liquid-like morphogenetic cell and tissue rearrangements of cell sorting, tissue spreading and segregation represent self-assembly processes guided by the diminution of adhesive-free energy as cells tend to maximize their mutual binding. This conclusion relates to the physics governing these morphogenetic phenomena and applies independently of issues such as the specificities of intercellular adhesives.


Assuntos
Adesão Celular/fisiologia , Modelos Biológicos , Morfogênese/fisiologia , Animais , Fenômenos Biofísicos , Biofísica , Caderinas/genética , Caderinas/metabolismo , Agregação Celular , Expressão Gênica , Células L , Camundongos , Tensão Superficial , Termodinâmica , Transfecção
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