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Pericellular Hyaluronan Coat Visualized in Live Cells With a Fluorescent Probe Is Scaffolded by Plasma Membrane Protrusions

Kirsi Rilla, Riikka Tiihonen, Anne Kultti, Markku Tammi and Raija Tammi

Institute of Biomedicine/Anatomy, University of Kuopio, Kuopio, Finland

Correspondence to: Kirsi Rilla, Institute of Biomedicine/Anatomy, University of Kuopio, PO Box 1627, FIN-70211 Kuopio, Finland. E-mail: kirsi.rilla{at}uku.fi

Many cell types wear up to 20-µm-wide hyaluronidase-sensitive surface coats, detected by exclusion of sedimenting particles like fixed erythrocytes. The structure of the coat is enigmatic, being apparently too thick to be accounted by random coils or even extended chains of just hyaluronan attached to cell surface. We have shown that hyaluronan synthesis enforced by green fluorescent protein–hyaluronan synthase transfection creates microvillous protrusions. The idea that the plasma membrane protrusions rather than hyaluronan alone is responsible for the exclusion space was studied with a fluorescent probe for hyaluronan and a dye with membrane affinity, applied to live cell cultures. Mesothelial and smooth muscle cells, fibroblasts, and chondrocytes, all known for their endogenously active hyaluronan synthesis, showed hyaluronan-coated plasma membrane protrusions, barely visible in phase contrast microscopy. Treatment with hyaluronidase and inhibition of hyaluronan synthesis caused retraction of the protrusions unless they were attached to substratum. Hyaluronan and the exclusion space were reduced, but did not disappear, by purified hyaluronan hexasaccharides that compete with hyaluronan attached to CD44. The results suggest that slender plasma membrane protrusions are an inherent feature of hyaluronan coats, form their scaffold, and largely result from ongoing hyaluronan synthesis in their plasma membrane. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials. (J Histochem Cytochem 56:901–910, 2008)

Key Words: hyaluronan • protrusions • coat • CD44 • glycocalyx

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