Clathrin sheets and cytoskeletal podosomes on the cytoplasmic side of ventral membranes of cultured osteoclasts

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Using a cell shearing method combined with freeze-drying and rotary shadowing replicas after shearing cells open with a stream of buffer, we examined the ultrastructure of clathrin sheets and cytoskeletal podosomes of cultured osteoclasts. Further, to visualize the confocal images for podosomes and clathrin sheets of isolated osteoclasts, we stained F, G-actin, and clathrin. Optical sections from confocal z-scanning with F-, G-actin staining showed the podosomes to be rosette in shape. From xy-scanning the F-actin was distributed in the core of the podosomes as a dot, whereas G-actin was present in a ring-like fashion. An immunofluorescence staining for clathrin was seen at the paramarginal area and central part of osteoclasts. Both the clathrin sheets and cytoskeletal podosomes survived the shearing process. The cytoplasmic side of the ventral membranes of the osteoclasts was characterized by the presence of clathrin sheets displayed as pentagons or hexagons. Podosomes were composed of cytoskeletal framework mediating widespread association of the microfilaments with the cytoplasmic side of the membrane. Some microfilaments in podosomes seem to be closely associated with the ventral membrane via small particles, but others appeared to terminate directly on the membrane. Podosomes are well known to be a specific cell-substratum attachment apparatus for osteoclasts. Besides, the present result suggests that clathrin functions are essential for not only receptor-mediated vesicles trafficking but also cell attachment.