Dataset Information

Single-Molecule Imaging of Proteoglycans in the Pericellular Matrix.

ABSTRACT: 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.

SUBMITTER: Scrimgeour J

PROVIDER: S-EPMC5768515 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

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Single-Molecule Imaging of Proteoglycans in the Pericellular Matrix.

Scrimgeour Jan J McLane Louis T LT Chang Patrick S PS Curtis Jennifer E JE

Biophysical journal 20171105 11

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 ob ...[more]

PMID: 29102037

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Project description:BackgroundDaily physiologic loading causes fluctuations in hydration of the intervertebral disc (IVD); thus, the embedded cells experience cyclic alterations to their osmotic environment. These osmotic fluctuations have been described as a mechanism linking mechanics and biology, and have previously been shown to promote biosynthesis in chondrocytes. However, this phenomenon has yet to be fully interrogated in the IVD. Additionally, the specialized extracellular matrix surrounding the cells, the pericellular matrix (PCM), transduces the biophysical signals that cells ultimately experience. While it is known that the PCM is altered in disc degeneration, whether it disrupts normal osmotic mechanotransduction has yet to be determined. Thus, our objectives were to assess: (1) whether dynamic osmotic conditions stimulate biosynthesis in nucleus pulposus cells, and (2) whether pericellular heparan sulfate proteoglycans (HSPGs) modulate the biosynthetic response to osmotic loading.MethodsBovine nucleus pulposus cells isolated with retained PCM were encapsulated in 1.5% alginate beads and treated with or without heparinase III, an enzyme that degrades the pericellular HSPGs. Beads were subjected to 1 h of daily iso-osmotic, hyper-osmotic, or hypo-osmotic loading for 1, 2, or 4 weeks. At each timepoint the total amount of extracellular and pericellular sGAG/DNA were quantified. Additionally, whether osmotic loading triggered alterations to HSPG sulfation was assessed via immunohistochemistry for the heparan sulfate 6-O-sulfertransferase 1 (HS6ST1) enzyme.ResultsOsmotic loading significantly influenced sGAG/DNA accumulation with a hyper-osmotic change promoting the greatest sGAG/DNA accumulation in the pericellular region compared with iso-osmotic conditions. Heparanase-III treatment significantly reduced extracellular sGAG/DNA but pericellular sGAG was not affected. HS6ST1 expression was not affected by osmotic loading.ConclusionResults suggest that hyper-osmotic loading promotes matrix synthesis and that modifications to HSPGs directly influence the metabolic responses of cells to osmotic fluctuations. Collectively, results suggest degeneration-associated modifications to pericellular HSPGs may contribute to the altered mechanobiology observed in disease.

Dataset Information

Single-Molecule Imaging of Proteoglycans in the Pericellular Matrix.

Publications

Single-Molecule Imaging of Proteoglycans in the Pericellular Matrix.

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