Journal of Electron Microscopy Advance Access originally published online on June 14, 2006
Journal of Electron Microscopy 2006 55(2):53-61; doi:10.1093/jmicro/dfl012
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The ruffled border and attachment regions of the apposing membrane of resorbing osteoclasts as visualized from the cytoplasmic face of the membrane
Asahi University School of Dentistry, Department of Anatomy Hozumi 1851, Mizuho, Gifu 501-0296, Japan
*To whom correspondence should be addressed. E-mail: takisaka{at}dent.asahi-u.ac.jp
The aim of our present research was to visualize how the plasma membrane is modified and how the cytoskeleton interacts with the attachment and ruffled border regions of resorbing osteoclasts. In order to view the surface modification of membranes and associated cytoskeleton, we employed the method of cell-shearing combined with quick-freezing and rotary replication to expose and replicate an extensive area of the cytoplasmic face of the surface membrane of osteoclasts in contact with synthetic apatite as a substratum. The membrane apposed to the apatite was composed of three different domains: the attachment zone, ruffled border and the remainder. In the attachment zone, a highly organized actin filament network formed dot-shaped, F-actin rich adhesion sites, so-called podosomes, and the actin ring. The cytoskeletal filament of podosomes and actin ring appeared to be in direct contact with the cytoplasmic surface of the underlying membrane. Within the actin ring, individually recognizable podosomes were well preserved, which indicates that the actin ring was probably derived from the fusion of podosomes. After shearing at the ruffled border region, the ruffled border projections and membrane regions among the projections were left behind. These ruffled border projections contained the cytoskeletal network. These actin networks also appeared to be in direct contact with the inner side of the ruffled border membrane or in contact with it via membrane-associated particles. At the basal portion of the ruffled border, numerous clathrin-coated patches or pits were well preserved. Deeper clathrin-coated pits and vesicles were also found, which indicates an active site for receptor-mediated endocytotic events. Clathrin sheets were also observed in the cell periphery outside of the actin ring. This type of clathrin sheets adhered to the apatite substrate, but was not anchored to the actin microfilaments. Our study thus clearly visualized the interaction between the cytoskeletal filaments and the underlying membrane at the ruffled border, attachment zone and podosome in osteoclasts cultured on apatitepellets.
Keywords osteoclast, actin ring, podosome, ruffled border, cell-shearing, quick-freezing
Received 27 December 2005, accepted 10 April 2006