RESUMEN
Interactions with the extracellular matrix (ECM) dictate cell fates. However, the complexity of dense ECM network and cell-surface molecules prevent the study of their dynamic interaction at the molecular level on living cells. Here, we focus on peptidyl prolyl cis/trans isomerases (PPIases) to dissect prolyl isomerization from other dynamic events. We reveal the contribution of PPIase on the mechanical properties of various ECM materials and on the dynamic cell-ECM interaction. To avoid complications associated with the existing spectroscopy-based methods such as light scattering, an assay was developed for detecting PPIase activity on living cell surface. This assay allows us to correlate PPIase activity with ECM development, and with the physiological and pathological states of the cells, including the functional properties of cancer cells and immune effector cells.
Asunto(s)
Ciclofilina A/metabolismo , Ciclofilinas/metabolismo , Matriz Extracelular/enzimología , Peptidilprolil Isomerasa de Interacción con NIMA/metabolismo , Proteínas de Unión a Tacrolimus/metabolismo , Membrana Celular/química , Membrana Celular/efectos de los fármacos , Membrana Celular/enzimología , Clonación Molecular , Ciclofilina A/genética , Ciclofilina A/farmacología , Ciclofilinas/genética , Ciclofilinas/farmacología , Ciclosporina/farmacología , Pruebas de Enzimas , Escherichia coli/genética , Escherichia coli/metabolismo , Matriz Extracelular/química , Matriz Extracelular/efectos de los fármacos , Fibrina/metabolismo , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/enzimología , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Humanos , Hidrogeles , Células Jurkat , Cinética , Peptidilprolil Isomerasa de Interacción con NIMA/genética , Peptidilprolil Isomerasa de Interacción con NIMA/farmacología , Cultivo Primario de Células , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Proteínas de Unión a Tacrolimus/genética , Proteínas de Unión a Tacrolimus/farmacologíaRESUMEN
Combinatorial screening represents a promising strategy to discover biomaterials for tailored cell culture applications. Although libraries incorporating different biochemical cues have been investigated, few simultaneously recapitulate relevant biochemical, physical, and dynamic features of the extracellular matrix (ECM). Here, a noncovalent system based on liquid-liquid phase separation (coacervation) and gelation mediated by glycosaminoglycan (GAG)-peptide interactions is reported. Multiple biomaterial libraries are generated using combinations of sulfated glycosaminoglycans and poly(ethylene glycol)-conjugated peptides. Screening these biomaterials reveals preferred biomatrices for the attachment of six cell types, including primary mesenchymal stromal cells (MSCs) and primary neural precursor cells (NPCs). Incorporation of GAGs sustains the expansion of all tested cell types comparable to standard cell culture surfaces, while osteogenic differentiation of MSC and neuronal differentiation of NPC are promoted on chondroitin and heparan biomatrices, respectively. The presented noncovalent system provides a powerful tool for developing tissue-specific ECM mimics.
Asunto(s)
Diferenciación Celular , Técnicas de Cultivo de Célula , Células Cultivadas , Matriz Extracelular , Células Madre Mesenquimatosas , Células-Madre Neurales , OsteogénesisRESUMEN
The pancreatic islet, a cellular community harboring the insulin-producing beta-cells, is known to undergo age-related alterations. However, only a handful of signals associated with aging have been identified. By comparing beta-cells from younger and older zebrafish, here we show that the aging islets exhibit signs of chronic inflammation. These include recruitment of tnfα-expressing macrophages and the activation of NF-kB signaling in beta-cells. Using a transgenic reporter, we show that NF-kB activity is undetectable in juvenile beta-cells, whereas cells from older fish exhibit heterogeneous NF-kB activity. We link this heterogeneity to differences in gene expression and proliferation. Beta-cells with high NF-kB signaling proliferate significantly less compared to their neighbors with low activity. The NF-kB signalinghi cells also exhibit premature upregulation of socs2, an age-related gene that inhibits beta-cell proliferation. Together, our results show that NF-kB activity marks the asynchronous decline in beta-cell proliferation with advancing age.
Asunto(s)
Envejecimiento , Proliferación Celular , Mediadores de Inflamación/metabolismo , Inflamación/patología , Células Secretoras de Insulina/patología , FN-kappa B/metabolismo , Pez Cebra/fisiología , Animales , Animales Modificados Genéticamente , Células Cultivadas , Perfilación de la Expresión Génica , Inflamación/inmunología , Inflamación/metabolismo , Células Secretoras de Insulina/inmunología , Células Secretoras de Insulina/metabolismo , FN-kappa B/genética , Transducción de Señal , Análisis de la Célula Individual , Activación Transcripcional , Pez Cebra/inmunologíaRESUMEN
The proliferative and functional heterogeneity among seemingly uniform cells is a universal phenomenon. Identifying the underlying factors requires single-cell analysis of function and proliferation. Here we show that the pancreatic beta-cells in zebrafish exhibit different growth-promoting and functional properties, which in part reflect differences in the time elapsed since birth of the cells. Calcium imaging shows that the beta-cells in the embryonic islet become functional during early zebrafish development. At later stages, younger beta-cells join the islet following differentiation from post-embryonic progenitors. Notably, the older and younger beta-cells occupy different regions within the islet, which generates topological asymmetries in glucose responsiveness and proliferation. Specifically, the older beta-cells exhibit robust glucose responsiveness, whereas younger beta-cells are more proliferative but less functional. As the islet approaches its mature state, heterogeneity diminishes and beta-cells synchronize function and proliferation. Our work illustrates a dynamic model of heterogeneity based on evolving proliferative and functional beta-cell states.Βeta-cells have recently been shown to be heterogeneous with regard to morphology and function. Here, the authors show that ß-cells in zebrafish switch from proliferative to functional states with increasing time since ß-cell birth, leading to functional and proliferative heterogeneity.
Asunto(s)
Células Secretoras de Insulina/citología , Islotes Pancreáticos/citología , Pez Cebra/embriología , Animales , Animales Modificados Genéticamente , Linaje de la Célula , Proliferación Celular , Técnicas Citológicas/métodos , Embrión no Mamífero/citología , Embrión no Mamífero/efectos de los fármacos , Glucosa/metabolismo , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/fisiología , Islotes Pancreáticos/embriología , Tamoxifeno/análogos & derivados , Tamoxifeno/farmacología , Urocortinas/metabolismo , Pez Cebra/genéticaRESUMEN
The proficiency of MTX loaded BSA capped gold nanoparticles (Au-BSA-MTX) in inhibiting the proliferation of breast cancer cells MCF-7 as compared to the free drug Methotrexate (MTX) is demonstrated based on MTT and Ki-67 proliferation assays. In addition, DNA ladder gel electrophoresis studies, flow cytometry and TUNEL assay confirmed the induction of apoptosis by MTX and Au-BSA-MTX in MCF-7 cells. Notably, Au-BSA-MTX was found to have higher cytotoxicity on MCF-7 cells compared with an equivalent dose of free MTX. The enhanced activity is attributed to the preferential uptake of Au-BSA-MTX particles by MCF-7 cells due to the presence of BSA that acts as a source of nutrient and energy to the rapidly proliferating MCF-7 cells. Moreover, the targeting ability of the drug MTX to the over expressed folate receptors on MCF-7 cells also contributes to the enhanced uptake and activity. Taken together, these results unveil that Au-BSA-MTX could be more effective than free drug for cancer treatment.
Asunto(s)
Antineoplásicos/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Portadores de Fármacos/química , Oro/química , Nanopartículas del Metal/química , Metotrexato/uso terapéutico , Albúmina Sérica Bovina/química , Caspasa 3/metabolismo , Ciclo Celular/efectos de los fármacos , Electroforesis en Gel de Agar , Femenino , Humanos , Antígeno Ki-67/metabolismo , Células MCF-7 , Nanopartículas del Metal/ultraestructura , Metotrexato/toxicidad , Espectrofotometría Atómica , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
Reduction of complexity of the extracellular matrix (ECM) to a non-covalent structure with minimal chemically defined components represents an attractive avenue for understanding the biology of the ECM. The resulting system could lead to the design of tailor-made biomaterials that incorporate varying functionalities. Negatively charged glycosaminoglycans are the major components of the ECM. Their interaction with positively charged proteins is important for dynamic three-dimensional scaffold formation and function. We designed and screened minimal peptide motifs whose conjugates with polyethylene glycol interact with heparin to form non-covalent hydrogels. Here we show the structure/function relationship of the (RA)(n) and (KA)(n) motifs and determined that both basic residues and the heparin-induced α-helix formation are important for the assembly process. Simple rules allowed us to tune various aspects of the matrix system such as the gelation rates, biodegradability, rheological properties, and biofunctionality. The hydrogels can encapsulate cells and support cell survival.
Asunto(s)
Matriz Extracelular/química , Heparina/química , Hidrogeles , Péptidos/química , Secuencia de Aminoácidos , Datos de Secuencia MolecularRESUMEN
A simple and convenient one step room temperature method is described for the synthesis of bovine serum albumin (BSA) capped gold and silver nanoparticles. BSA reduces silver ions to silver nanoparticles but does not directly reduce gold ions to gold nanoparticles at room temperature and varying pH conditions. However, when silver and gold ions are simultaneously added to BSA, silver ions get reduced to metallic silver first and these in turn reduce gold ions to gold nanoparticles through a galvanic exchange reaction. The so synthesized silver and gold nanoparticles are easily water dispersible and can withstand addition of salt even at high concentrations. It is shown that the capped protein retains its secondary structure and the helicity to a large extent on the nanoparticles surface and that the protein capping makes the nanoparticles cytocompatible.