Search

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.

Conventional studies of dynamic phagocytic behavior have been limited in terms of spatial and temporal resolution due to the inherent three-dimensionality and small features of phagocytosis. To overcome these issues, we use a series of frustrated phagocytosis assays to quantitatively characterize phagocytic spreading dynamics. Our investigation reveals that frustrated phagocytic spreading occurs in phases and is punctuated by a distinct period of contraction. The spreading duration and peak contact areas are independent of the surface opsonin density, although the opsonin density does affect the likelihood that a cell will spread. This reinforces the idea that phagocytosis dynamics are primarily dictated by cytoskeletal activity. Structured illumination microscopy reveals that F-actin is reorganized during the course of frustrated phagocytosis. F-actin in early stages is consistent with that observed in lamellipodial protrusions. During the contraction phase, it is bundled into fibers that surround the cell and is reminiscent of a contractile belt. Using traction force microscopy, we show that cells exert significant strain on the underlying substrate during the contraction phase but little strain during the spreading phase, demonstrating that phagocytes actively constrict during late-stage phagocytosis. We also find that late-stage contraction initiates after the cell surface area increases by 225%, which is consistent with the point at which cortical tension begins to rise. Moreover, reducing tension by exposing cells to hypertonic buffer shifts the onset of contraction to occur in larger contact areas. Together, these findings provide further evidence that tension plays a significant role in signaling late-stage phagocytic activity.

We present a detailed characterization of the organization of hyaluronan and bottlebrush proteoglycans in the pericellular matrix (PCM) of rat chondrocyte cells (RCJ-P) and PC3 prostrate cancer cells, and then use that insight to understand how the PCM impacts the transport of nanoparticles and charged molecules to the cell surface, and the cell's adhesion to substrates. Our studies reveal that the PCM strongly impacts the flux of nanoparticles to the cell surface in a size-dependent fashion. Further, the concentration of bottlebrushes (e.g. aggrecan, versican) in the PCM determines a maximal particle size that can reach the cell surface, ∼400nm for both cell lines but only ∼200nm when the PCM is saturated with proteoglycans. These observations have direct implications for studies of drug delivery vehicles and exosomes, whose particle sizes fall in this range. Our studies also provide evidence that negatively-charged chondroitin sulfates in the bottlebrush proteoglycans are available for binding to positively-charged molecules (e.g. growth factors), sequestering and concentrating molecules near the cell surface, as well as protecting them from enzymatic degradation. Super-resolution imaging of PCM at the cell-substrate interface reveals patches of PCM in co-existence with vinculin-rich adhesions. In order to address whether the PCM acts as a repulsive cushion, we quantify the cell adhesion strength versus enzymatic degradation of the PCM using a spinning disk apparatus. The studies show that the adhesion strength increases by ∼24% for RCJP cells enzymatically-treated with hyaluronidase and by a factor of ∼39% for PC3 cells. A similar increase in adhesion occurs with chondroitinase-ABC treatment. Together, these quantitative measurements of the pericellular matrix indicate that this common but neglected in vitro and in vivo structure deserves further attention to understand how it regulates cells’ interactions with their surroundings. Funding: NSF CAREER-DMR-0848797.

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.