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1.
Biophys J ; 113(11): 2316-2320, 2017 Dec 05.
Artículo en Inglés | MEDLINE | ID: mdl-29102037

RESUMEN

The pericellular matrix is a robust, hyaluronan-rich polymer brush-like structure that controls access to the cell surface, and plays an important role in cell adhesion, migration, and proliferation. We report the observation of single bottlebrush proteoglycan dynamics in the pericellular matrix of living chondrocytes. Our investigations show that the pericellular matrix undergoes gross extension on the addition of exogenous aggrecan, and that this extension is significantly in excess of that observed in traditional particle exclusion assays. The mean-square displacement of single, bound proteoglycans increases with distance to cell surface, indicating reduced confinement by neighboring hyaluronan-aggrecan complexes. This is consistent with published data from quantitative particle exclusion assays that show openings in the pericellular matrix microstructure ranging from ∼150 nm near the cell surface to ∼400 nm near the cell edge. In addition, the mobility of tethered aggrecan drops significantly when the cell coat is enriched with bottlebrush proteoglycans. Single-molecule imaging in this thick polysaccharide matrix on living cells has significant promise in the drive to elucidate the role of the pericellular coat in human health.


Asunto(s)
Imagen Molecular , Proteoglicanos/metabolismo , Análisis de la Célula Individual , Animales , Membrana Celular/metabolismo , Condrocitos/citología , Ratas
2.
Biophys J ; 110(12): 2739-2750, 2016 Jun 21.
Artículo en Inglés | MEDLINE | ID: mdl-27332132

RESUMEN

The hyaluronan-rich pericellular matrix (PCM) plays physical and chemical roles in biological processes ranging from brain plasticity, to adhesion-dependent phenomena such as cell migration, to the onset of cancer. This study investigates how the spatial distribution of the large negatively charged bottlebrush proteoglycan, aggrecan, impacts PCM morphology and cell surface access. The highly localized pericellular milieu limits transport of nanoparticles in a size-dependent fashion and sequesters positively charged molecules on the highly sulfated side chains of aggrecan. Both rat chondrocyte and human mesenchymal stem cell PCMs possess many unused binding sites for aggrecan, showing a 2.5x increase in PCM thickness from ∼7 to ∼18 µm when provided exogenous aggrecan. Yet, full extension of the PCM occurs well below aggrecan saturation. Hence, cells equipped with hyaluronan-rich PCM can in principle manipulate surface accessibility or sequestration of molecules by tuning the bottlebrush proteoglycan content to alter PCM porosity and the number of electrostatic binding sites.


Asunto(s)
Agrecanos/metabolismo , Matriz Extracelular/metabolismo , Animales , Proteínas Bacterianas/metabolismo , Membrana Celular/metabolismo , Células Cultivadas , Condroitina ABC Liasa/metabolismo , Difusión , Matriz Extracelular/microbiología , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Ácido Hialurónico/metabolismo , Células Madre Mesenquimatosas/metabolismo , Nanopartículas/metabolismo , Pinzas Ópticas , Porosidad , Proteus vulgaris , Ratas , Electricidad Estática
3.
Biophys J ; 104(5): 986-96, 2013 Mar 05.
Artículo en Inglés | MEDLINE | ID: mdl-23473481

RESUMEN

A voluminous polymer coat adorns the surface of many eukaryotic cells. Although the pericellular matrix (PCM) often extends several microns from the cell surface, its macromolecular structure remains elusive. This massive cellular organelle negotiates the cell's interaction with surrounding tissue, influencing important processes such as cell adhesion, mitosis, locomotion, molecular sequestration, and mechanotransduction. Investigations of the PCM's architecture and function have been hampered by the difficulty of visualizing this invisible hydrated structure without disrupting its integrity. In this work, we establish several assays to noninvasively measure the ultrastructure of the PCM. Optical force probe assays show that the PCM of rat chondrocyte joint (RCJ-P) cells easily reconfigures around optically manipulated microparticles, allowing the probes to penetrate into rather than compress the matrix. We report distinct changes in forces measured from PCMs treated with exogenous aggrecan, illustrating the assay's potential to probe proteoglycan distribution. Measurements reveal an exponentially increasing osmotic force in the PCM arising from an inherent concentration gradient. With this result, we estimate the variation of the PCM's mesh size (correlation length) to range from ∼100 nm at the surface to 500 nm at its periphery. Quantitative particle exclusion assays confirm this prediction and show that the PCM acts like a sieve. These assays provide a much-needed tool to study PCM ultrastructure and its poorly defined but important role in fundamental cellular processes.


Asunto(s)
Condrocitos/citología , Matriz Extracelular/ultraestructura , Pinzas Ópticas , Agrecanos/farmacología , Animales , Línea Celular , Matriz Extracelular/efectos de los fármacos , Concentración Osmolar , Ratas
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