Ultrastructural analysis of nanogold-labeled cell surface microvilli in liquid by atmospheric scanning electron microscopy and their relevance in cell adhesion

Abstract

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.

Figure 1

Correlative light and electron microscopy (CLEM) by atmospheric scanning electron microscope (ASEM). (A) ASEM dish. A removable 35-mm ASEM dish has a 100-nm silicon nitride (SiN) film window separating vacuum and atmosphere; (B) SEM image of an SiN window. Scale bar, 500 μm; and (C) Schematic diagram of the ASEM system. An optical microscope capable of fluorescence imaging, is arranged above the inverted SEM, with the specimen dish between them. The removable 35-mm ASEM dish features an SiN film window in its bottom plate, which separates vacuum and atmosphere. The electron beam is projected from underneath onto the cells through the SiN film, and backscattered electrons are captured by the backscattered electron imaging (BEI) detector. The axes of both microscopes are mechanically aligned, and the specimen stage can be shifted two-dimensionally on the X–Y plane.

Figure 2

Distribution of CD44 on the plasma membrane, visualized by correlative optical and electron microscopy using ASEM. BW5147 T lymphocytes were labeled with an anti-CD44 monoclonal antibody IM7.8.1, and further with a secondary antibody conjugated both with Alexa Fluor 488 and Nanogold. (A) Fluorescence optical microscopy (OM) image; and (B) Electron microscopy (EM) image of the same cells after gold enhancement. Signals were clearly observed on the cell body as well as on the microvilli. ASEM images were captured at 3500× magnification. Scale bar represents 5 μm.

Figure 3

The effect of cytochalasin D on CD44 distribution and activity. (A) BW5147 T lymphocytes were left untreated (left panels) or treated with 10 μM cytochalasin D on ASEM dishes for 1 h (right panels), and fixed with 4% paraformaldehyde. CD44 on the cell surface was labeled with FluorNanogold. The labeled cells were observed by fluorescence microscopy (upper panels) or electron microscopy (lower panels) after gold enhancement using ASEM at 4000× (lower left panel) and 5500× (lower right panel) magnifications. Scale bars represent 5 μm (left panel) and 2 μm (right panel); and (B) The effect of cytochalasin D on hyaluronan-binding ability. BW5147 T lymphocytes were treated with 10 μM cytochalasin D (thick line) or left untreated (thin line) for 1 h at 37 °C, and the extent of FITC-conjugated hyaluronan binding was determined by flow cytometry. Gray filled profile, unstained control.

Figure 3

The effect of cytochalasin D on CD44 distribution and activity. (A) BW5147 T lymphocytes were left untreated (left panels) or treated with 10 μM cytochalasin D on ASEM dishes for 1 h (right panels), and fixed with 4% paraformaldehyde. CD44 on the cell surface was labeled with FluorNanogold. The labeled cells were observed by fluorescence microscopy (upper panels) or electron microscopy (lower panels) after gold enhancement using ASEM at 4000× (lower left panel) and 5500× (lower right panel) magnifications. Scale bars represent 5 μm (left panel) and 2 μm (right panel); and (B) The effect of cytochalasin D on hyaluronan-binding ability. BW5147 T lymphocytes were treated with 10 μM cytochalasin D (thick line) or left untreated (thin line) for 1 h at 37 °C, and the extent of FITC-conjugated hyaluronan binding was determined by flow cytometry. Gray filled profile, unstained control.

Figure 4

ASEM images by positively charged Nanogold labeling. (A) Cells cultured on the ASEM dish were fixed, stained with positively charged Nanogold, and treated with GoldEnhance-EM. 3500× magnification. Scale bar, 5 μm; and (B) The cell body was further magnified at 8000× magnification. Scale bar, 2 μm.

Figure 5

Disruption of the cytoskeletal structure with cytochalasin D abrogates CD44-mediated rolling adhesion under flow conditions. BW5147 T lymphocytes were left untreated or treated with cytochalasin D (10 μM for 1 h), and were then applied continuously to capillary tubes whose inner surface had been coated with hyaluronan. The number of rolling cells at wall shear stress of 1.2 dyn/cm² was determined as described in the Experimental Section.