Direct visualization of epithelial microvilli biogenesis

Abstract

Microvilli are actin bundle supported surface protrusions that play essential roles in diverse epithelial cell functions. To develop our understanding of microvilli biogenesis, we used live imaging to directly visualize protrusion growth at early stages of epithelial differentiation. Time-lapse data revealed that an 'initiation complex' enriched in EPS8 and IRTKS appears at future sites of microvillus growth minutes before core actin bundle assembly. Elongation of a new core bundle occurs in parallel with the arrival of EZRIN and plasma membrane encapsulation. In addition to de novo growth, we also observed that new microvilli emerge from pre-existing protrusions. Additionally, we found that new microvilli can also collapse, characterized first by loss of membrane wrapping and Ezrin enrichment, followed by a sharp decrease in distal tip EPS8 and IRTKS. These studies are the first to offer a temporally resolved microvillus growth mechanism and highlight critical factors that drive this process.

High resolution dynamic mapping of the C. elegans intestinal brush border

“…Interestingly, we observed that FLN-2 displayed a shifted pattern, with an earlier apical accumulation that may suggest a specific role in microvilli establishment that needs to be analysed in detail. Thus, as in mammalian cells (Gaeta et al, 2021), C. elegans microvilli assembly might rely on an initiation complex, composed, at least, of ERM-1, ACT-5, PLST-1, FLN-2 and IFB-2, and an elongation/maturation complex, composed additionally of the actin polymerization/severing agent EPS-8, HUM-5 and MLC-5 (Fig. S5B).…”

“…Expression profiling in mammalian enterocytes between the proliferative crypt and the terminally differentiated villus demonstrated a marked upregulation of actin cytoskeleton-related genes, including actin, ezrin, villin and espin (Chang et al, 2008, Mariadason et al, 2005). Notably, recent data in LLC-PKI cells showed a stepwise recruitment of EPS8 and IRTKS first, and then ezrin, during microvilli outgrowth (Gaeta et al, 2021). We hypothesized that a set of brush border components may specifically be recruited at the apical pole during brush border establishment in vivo.…”

“…Expression profiling in mammalian enterocytes between the proliferative crypt and the terminally differentiated villus demonstrated a marked upregulation of actin-related cytoskeletal genes, including actin, ezrin, villin and espin (Chang et al, 2008, Mariadason et al, 2005. Notably, recent data in LLC-PK1 cells showed a stepwise recruitment of EPS8 and IRTKS before (initiation) and ezrin during (elongation) microvilli growth (Gaeta et al, 2021).…”

“…Recent studies in epithelial cell lines identified new functional players, such as IRTKS or myosin la/6/7b (Crawley et al, 2014a, Postema et al, 2018), and new mechanisms of brush border assembly and maintenance by vesicular trafficking (Vogel et al, 2015), microvilli motility, contraction and clustering (Meenderink et al, 2019, Chinowsky et al, 2020) or intermicrovillar protocadherin bridges (Crawley et al, 2014b). Additionally, recent use of live imaging revealed some of the key initiation and maturation steps of microvilli biogenesis in cell lines (Gaeta et al, 2021). However, a description of brush border formation and maintenance in vivo is lacking.…”

Mitotic Spindle Positioning (MISP) is an actin bundler that selectively stabilizes the rootlets of epithelial microvilli

“…This kinetic difference at opposite ends creates a system that allows subunits to flux retrograde through the core bundle in a process referred to as ‘treadmilling’ (Kirschner, 1980). Indeed, recent studies with epithelial cell culture models revealed that treadmilling is crucial for microvilli assembly and motility (Gaeta et al, 2021; Meenderink et al, 2019).…”