The microvilli or ‘brush border’ of intestinal and kidney epithelial cells has been an intense area of research and focussed on actin filament organization and associated molecular interactions. The presence of microvilli on epithelial cells is dependent on a network of intricately synchronized pathways and, at least in part, members of the ERM (ezrin/radixin/moesin) protein family1. The best characterized ERM in mammalian cells, often used to study the brush border, such as intestinal Caco-2 and kidney MDCK cells is ezrin and is itself associated with EBP50/NHERF1 when active2. This association is required for microvilli formation. In addition the serine/threonine kinase SLK1 is required to keep active ezrin and thus microvilli assembly correctly positioned3.
To study the molecular mechanism of microvilli assembly and thus related actin filament reorganization, microvilli structure must be measured in a quantitative manner as an assay to gauge various molecular manipulations in such studies. Current protocols involve using TEM to analyse the ultrastructure of microvilli and imaging analysis software to measure the length of microvilli4. Since analysis of cut cell or tissue sections are generally around 50-70 nm thick, an unrealistic number of ultrathin sections would need to be prepared to reconstruct the entire apical surface of analysed specimens at the ultrastructural level, which is both too time consuming and expensive as a routine assay. In this protocol we outline an approach that is relatively economical both with regards to time and financial cost and robust as a routine assay. We describe sample preparation of analysed cells for scanning electron microscopy (SEM) analysis, the analysis of cell surface and processing of the raw data using Nikon Imaging Software (NIS) that enables the identification of microvilli ultrastructure formations that range from ridge-like protrusions, single buds to more advanced formations that are referred to, here, collectively as microvilli clusters and measured by area (see Figure 1). Hence, from the initial molecular experimentation of cellular processes to final microvilli analysis the current protocol can correlate molecular mechanism to the degree of microvilli assembly, quantitatively with corresponding ultrastructural imaging of the entire apical surface of epithelial cells as an effective assay tool.