Chikara Sato

Ultrastructural Analysis of Nanogold-Labeled Cell Surface Microvilli in Liquid by Atmospheric Scanning Electron Microscopy and Their Relevance in Cell Adhesion

The adhesion of leukocytes circulating in the blood to vascular endothelium is critical for their trafficking in the vasculature, and CD44 is an important cell surface receptor for rolling adhesion. In this study, we demonstrate the correlative observation of CD44 distribution at the lymphocyte cell surface in liquid by fluorescence optical microscopy and immuno-electron microscopy using an atmospheric scanning electron microscope (ASEM). The ultrastructure of the cell surface was clearly imaged by ASEM using positively charged Nanogold particles. ASEM analysis demonstrated microvilli projections around the cell surface and the localization of CD44 on the microvilli. Treatment of cells with cytochalasin D resulted in a loss of the microvilli projections and concomitantly abrogated CD44-mediated adhesion to its ligand hyaluronan. These results suggest the functional relevance of microvilli in CD44-mediated rolling adhesion under shear flow.

Assembly of protein complexes restricts diffusion of Wnt3a proteins

Members of the Wnt protein family play roles in many aspects of embryogenesis and homeostasis. Despite their biological significance, characteristics of Wnt proteins still remain unclear, mainly due to their insolubility after the removal of serum. Here we examine Wnt proteins in serum-containing media by using analytical ultracentrifugation with a fluorescence detection system. This analysis reveals that Wnt3a assembles into high-molecular-weight complexes that become dissociable by interaction with the extracellular domain of the Frizzled8 receptor or secreted Wnt-binding protein sFRP2. Cross-linking and single-particle analyses of Wnt3a fractionated by gel filtration chromatography show the homo-trimer to be the smallest form of the assembled Wnt3a complexes. Fluorescence correlation spectroscopy and immunohistochemistry reveal that the assembly of Wnt3a complexes restricted their diffusion and signaling range in Xenopus laevis embryos. Thus, we propose that the Wnt diffusion range can be controlled by a balance between the assembly of Wnt complexes and their dissociation.Ritsuko Takada et al. show that Wnt3a assembles into high molecular weight complexes that restrict the diffusion of Wnt within Xenopus embryos. These results suggest that Wnt diffusion in cells is controlled by a balance between higher order complex assembly and dissociation by Wnt-binding proteins.