Intraepidermal cell surface fine structure: preservation and examination at high resolution

Investigating cell surfaces in solid tissues has been challenging due to damage caused by traditional separation methods. These techniques, such as mechanical or enzymatic dispersion, often compromise the integrity of the cell surfaces. Prior research has shown that cultured cells are more commonly used for such studies because they avoid this issue. However, this approach limits the ability to study native tissue structures. The need for a method that preserves cell surface morphology without causing damage has remained unmet. This gap motivated the search for alternative techniques that could maintain the ultrastructural details of solid tissues. The development of such methods is essential for understanding the true topography of cell surfaces in their natural context. No prior work had resolved the issue of preserving intercellular junctions in intact epithelia. The lack of suitable techniques hindered the ability to study desmosomes and other surface features in their native state. This uncertainty drove the exploration of new approaches to examine cell surfaces in solid tissues without structural compromise.

This study aimed to develop a method for examining cell surfaces in solid tissues without causing structural damage. The researchers focused on the intraepidermal cell surfaces of mouse and human squamous epithelia. They sought to avoid the limitations of mechanical, enzymatic, or chelator-based dispersal methods. The primary objective was to preserve the ultrastructural integrity of the cell surfaces during preparation. By using a specific toxin, the team aimed to cleave intercellular spaces without cytotoxic effects. This approach allowed for the study of native cell surface features in their undisturbed state. The goal was to enable high-resolution imaging of structures like desmosomes and microvilli. The study aimed to provide a reliable method for preserving and examining cell surface morphology in solid tissues.

The researchers used staphylococcal epidermolytic toxin to cleave intercellular spaces in mouse and human squamous epithelia. This toxin allowed for separation without causing ultrastructural damage. Neonatal mouse epidermis was collected two hours after toxin injection. Immediately after sacrifice, tissues were immersed in fixative to preserve intraepithelial surfaces. Specimens were prepared using freeze-fracture techniques for electron microscopy. Some samples were embedded for transmission electron microscopy. Critical-point drying was used before platinum/carbon replication for scanning and transmission electron microscopy. Replicas required stabilization with parloidion and cleaning with bleach and 40% chromate. These methods enabled the visualization of external membrane structures at high resolution.

The study revealed a convoluted cell surface covered with microvilli, 15–20 nm particles, and desmosomal mounds. Freeze-fracture and scanning electron microscopy showed tenuous microvilli projecting from the surface. Scattered 15–20 nm particles were observed across the cell surface. Hemispherical desmosomal mounds were identified as part of the surface topography. Desmosomal plaques contained 15–20 nm globular particles arranged randomly. These particles matched in distribution and density to those seen in freeze-fractured desmosomes. The findings suggest that desmosomal integral membrane particles span the external leaflet of the plasma membrane. The combination of four imaging methods allowed for the accurate identification of external membrane structures.

The authors propose that the staphylococcal epidermolytic toxin provides a non-cytotoxic method for separating intercellular spaces. This technique preserves the ultrastructural integrity of cell surfaces in solid tissues. The study demonstrates that freeze-fracture, scanning, and transmission electron microscopy can be used together to examine external membrane structures. The presence of 15–20 nm particles in desmosomal plaques suggests a transmembrane arrangement. The findings support the idea that these particles span the external leaflet of the plasma membrane. The use of parloidion stabilization and chromate cleaning was essential for preparing replicas. The combination of four complementary methods enabled the identification of surface features. The results suggest that this approach can be used to study cell surfaces in their native state without structural compromise.